Document Detail

Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.
MedLine Citation:
PMID:  23017083     Owner:  NLM     Status:  MEDLINE    
We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.
Alex J Hughes; Augusto M Tentori; Amy E Herr
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-10-10
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  134     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-24     Completed Date:  2013-03-28     Revised Date:  2014-11-02    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  17582-91     Citation Subset:  IM    
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MeSH Terms
Anthozoa / chemistry
Green Fluorescent Proteins / chemistry*,  genetics
In Situ Hybridization, Fluorescence*
Isoelectric Focusing*
Isoelectric Point
Microfluidic Analytical Techniques*
Photochemical Processes
Grant Support
Reg. No./Substance:
147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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